Dataset Information

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids

ABSTRACT: 3D spheroids of primary human hepatocytes (3D PHH) keep a differentiated phenotype with retained metabolic function and proteome fingerprint for weeks in culture. As a result, 3D PHH are gaining ground as a model for mechanistic studies on liver homeostasis and in vitro to in vivo (IVIV)predictions in drug discovery. However, the function of drug-transporting proteins has not yet been studied in the 3D PHH. Here we used the organic cation transporter 1 (OCT1/SLC22A1) as a model to explore both transporter kinetics and long-term regulation of transporter activity via different pathways. The 3D PHH were cultured for one week and then used for short- and long-term studies. The OCT1 transporter kinetics was assessed using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, the 3D PHH were exposed to xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomics analysis was used to track prototypical changes of other regulated proteins. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14.14±3.97 using three donors. Established OCT1 inhibitors reduced the uptake of ASP+ in the 3D PHH spheroids, resulting in 40-60% of the maximal uptake rate of ASP+. The long-term exposure studies showed that OCT1 is relatively stable to the activation of a broad range of pathways known to regulate ADME proteins. Importantly, while expression levels of other ADME proteins, such as CYP3A4 and MDR1 were regulated as expected, OCT1 levels remained stable. In conclusion, our results show for the first time that 3D PHH spheroids express fully active OCT1 and that transporter kinetics can be studied in 3D PHH. We also confirm that OCT1 remains stable and functional during activation of various pathways that alter the expression and function of other ADME proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Colon Cancer,Breast Cancer

SUBMITTER: Evgeniya Mickols

LAB HEAD: Per Artursson

PROVIDER: PXD045733 | Pride | 2024-08-10

REPOSITORIES: Pride

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Publications

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids.

Mickols Evgeniya E Meyer Alina A Handin Niklas N Stüwe Malin M Eriksson Jens J Rudfeldt Jakob J Blom Kristin K Fryknäs Mårten M Sellin Mikael E ME Lauschke Volker M VM Karlgren Maria M Artursson Per P

Scientific reports 20240727 1

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a mode ...[more]

PMID: 39068198

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Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

Dataset Information

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids

Publications

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids.

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